Activation Of Protein Kinase Research Articles

Kinome profiling identifies MARK3 and STK10 as potential therapeutic targets in uveal melanoma

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the “dark kinome”. Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

Maackiain suppresses the development of cervical cancer via AMPK priming autophagy.

Maackiain (Mac), a flavonoid analog isolated from Sophora flavescens, exhibits neuroprotective, anti-allergic, anti-inflammatory, and pro-apoptotic effects. It is not clear whether Mac has a therapeutic effect on cervical cancer. In this work, we used RT-qPCR, western blot, immunofluorescence, and related methods to detect the therapeutic mechanism of Mac for cervical cancer. We demonstrated that Mac significantly inhibited the proliferation, migration, and invasion of human cervical cancer cell lines HeLa and SiHa. And, Mac enhanced the pro-apoptotic effects of cisplatin in treating cervical cancer cells. Mac has shown good efficacy in treating cervical cancer. Furthermore, Mac inhibited the mammalian target of the rapamycin (mTOR) pathway, thereby inducing autophagy in cervical cancer cells. The regulation of mTOR/autophagy pathway by Mac relied on the activation of AMP-activated protein kinase (AMPK), and the inhibition of the AMPK reversed the Mac's anti-cervical cancer activity. In addition, experimental study of Mac in mouse xenograft tumor model further confirmed its good anti-cervical cancer activity. Mac inhibits human cervical cancer by activating the AMPK/mTOR/autophagy pathway, indicating that it is a potential natural compound for the treatment of cervical cancer. This study also provides a feasible molecular mechanism for the treatment of cervical cancer.

Generation of glucocorticoid-producing cells derived from human pluripotent stem cells

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, invitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency.

Mulberry leaf flavonoids activate BAT and induce browning of WAT to improve type 2 diabetes via regulating the AMPK/SIRT1/PGC-1α signaling pathway

Mulberry (Morus alba L.) leaf is a well-established traditional Chinese botanical and culinary resource. It has found widespread application in the management of diabetes. The bioactive constituents of mulberry leaf, specifically mulberry leaf flavonoids (MLFs), exhibit pronounced potential in the amelioration of type 2 diabetes (T2D). This potential is attributed to their ability to safeguard pancreatic β cells, enhance insulin resistance, and inhibit α-glucosidase activity. Our antecedent research findings underscore the substantial therapeutic efficacy of MLFs in treating T2D. However, the precise mechanistic underpinnings of MLF’s anti-T2D effects remain the subject of inquiry. Activation of brown/beige adipocytes is a novel and promising strategy for T2D treatment. In the present study, our primary objective was to elucidate the impact of MLFs on adipose tissue browning in db/db mice and 3T3-L1 cells and elucidate its underlying mechanism. The results manifested that MLFs reduced body weight and food intake, alleviated hepatic steatosis, improved insulin sensitivity, and increased lipolysis and thermogenesis in db/db mice. Moreover, MLFs activated brown adipose tissue (BAT) and induced the browning of inguinal white adipose tissue (IWAT) and 3T3-L1 adipocytes by increasing the expressions of brown adipocyte marker genes and proteins such as uncoupling protein 1 (UCP1) and beige adipocyte marker genes such as transmembrane protein 26 (Tmem26), thereby promoting mitochondrial biogenesis. Mechanistically, MLFs facilitated the activation of BAT and the induction of WAT browning to ameliorate T2D primarily through the activation of AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) signaling pathway. These findings highlight the unique capacity of MLF to counteract T2D by enhancing BAT activation and inducing browning of IWAT, thereby ameliorating glucose and lipid metabolism disorders. As such, MLFs emerge as a prospective and innovative browning agent for the treatment of T2D.

Metformin reduces hepatocarcinogenesis by inducing downregulation of Cyp26a1 and CD8+ T cells.

Hepatocellular carcinoma (HCC) is a highly heterogeneous cancer with major challenges in both prevention and therapy. Metformin, adenosine monophosphate-activated protein kinase (AMPK) activator, has been suggested to reduce the incidence of HCC when used for patients with diabetes in preclinical and clinical studies. However, the possible effects of metformin and their mechanisms of action in non-diabetic HCC have not been adequately investigated. Fah-/- mice were used to construct a liver-injury-induced non-diabetic HCC model for exploring hepatocarcinogenesis and therapeutic potential of metformin. Changes in relevant tumour and biochemical indicators were measured. Bulk and single-cell RNA-sequencing analyses were performed to validate the crucial role of proinflammatory/pro-tumour CD8+ T cells. In vitro and in vivo experiments were performed to confirm Cyp26a1-related antitumour mechanisms of metformin. RNA-sequencing analysis showed that chronic liver injury led to significant changes in AMPK-, glucose- and retinol metabolism-related pathways in Fah-/- mice. Metformin prevented the formation of non-diabetic HCC in Fah-/- mice with chronic liver injury. Cyp26a1 ddexpression in hepatocytes was significantly suppressed after metformin treatment. Moreover, downregulation of Cyp26a1 occurred in conjunction with increased levels of all-trans-retinoic acid (atRA), which is involved in the activation of metformin-suppressed hepatocarcinogenesis in Fah-/- mice. In contrast, both CD8+ T-cell infiltration and proinflammatory/pro-tumour cytokines in the liver were significantly upregulated in Fah-/- mice during chronic liver injury, which was notably reversed by either metformin or atRA treatment. Regarding mechanisms, metformin regulated the decrease in Cyp26a1 enzyme expression and increased atRA expression via the AMPK/STAT3/Gadd45β/JNK/c-Jun pathway. Metformin inhibits non-diabetic HCC by upregulating atRA levels and downregulating CD8+ T cells. This is the first reporting that the traditional drug metformin regulates the metabolite atRA via the Cyp26a1-involved pathway. The present study provides a potential application of metformin and atRA in non-diabetic HCC.

The SMAC mimetic birinapant alleviates lipopolysaccharide-induced acute lung injury by inhibiting MAPK signaling

The second mitochondria-derived activator of caspases (SMAC) mimetic birinapant attenuated liver injury by inhibited the degradation of tumor necrosis factor receptor-associated factor 3 (TRAF3) and activation of mitogen-activated protein kinase (MAPK) signaling pathway in liver macrophage, but its role in LPS induced acute lung injury (ALI) is not understood. The present study was to investigate the effects of birinapant on ALI and its possible mechanism. A dose of birinapant (30 mg/kg) or a vehicle was administered intravenously 24 hours before LPS (100 μg) stimulation in mice. The levels of TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The infiltrated macrophages and expression of monocyte chemoattractant protein-1 (MCP-1) was determined by immunohistochemistry staining in the lung tissues. The JNK and p38 MAPK activation, protein expression and K48-linked polyubiquitination of TRAF3 were determined in alveolar macrophage cell line (MH-S cells) after 1μg/ml LPS stimulation. The results showed that the birinapant down-regulated the levels of TNF-α, IL-6 and IL-1β in the BALF. In addition, birinapant markedly inhibited macrophages infiltration and MCP-1 protein expression in lung tissues. At last, birinapant suppressed the MAPKsignaling pathway and K48-linked ubiquitinated degradation of TRAF3 in MH-S cells after LPS administration. In conclusion, the results proved that birinapant protected against LPS-induced ALI through inhibiting MAPK activation and K48-linked ubiquitination of TRAF3 in alveolar macrophages.

Purified Polysaccharides Extracted from Gray Oyster Mushroom (Pleurotus sajor-caju (Fr.) Sing.) Stimulate Glucose Uptake in C2C12 Myotubes through the activation of AMP-Activated Protein Kinase (AMPK) and Glucose Transporter 1 (GLUT1) Proteins

The grey oyster mushroom [Pleurotus sajor-caju (Fr.) Sing.], an edible mushroom, has been known as a source of bioactive compounds, including polysaccharides. Polysaccharides from this mushroom have been shown to possess antidiabetic activity both in vivo and in vitro. However, antidiabetic mechanism of partially purified or purified polysaccharides from the gray oyster mushroom has not been characterized. In this study, we extracted and purified polysaccharides from gray oyster mushrooms and used them to investigate the antidiabetic mechanism in the context of C2C12 myotubes. Using Fourier Transform Infrared spectroscopy (FTIR) analysis and enzymatic assay, we showed that the polysaccharide sample, namely 9S1-1, contains b-glucose, a-glucose, and mannose as the monosaccharide composition, and b-glucan is the major type of polysaccharide in the sample. This 9S1-1 sample dose-dependently stimulated glucose uptake in C2C12 myotubes. Further analysis showed that the sample activated the AMP-activated protein kinase (AMPK) but not Akt serine/threonine kinase (AKT) phosphorylation, suggesting that the stimulation is AMPK-dependent. Moreover, we showed that compound c, an inhibitor of AMPK, inhibited glucose uptake in 9S1-1-stimulated cells, confirming the requirement of AMPK in the glucose uptake activated by the 9S1-1 sample. In addition, it promoted glucose transporter protein type 1 (GLUT1) but not GLUT4 protein expression. These results suggest that GLUT1 may be responsible for the stimulation of glucose uptake in 9S1-1-activated cells. Together, these data illustrate the antidiabetic mechanism of polysaccharides isolated from the gray oyster mushroom and the potential use of the polysaccharide as an antidiabetic agent.

Feedback enhanced tumor targeting delivery of albumin-based nanomedicine to amplify photodynamic therapy by regulating AMPK signaling and inhibiting GSTs

Oxidative therapies receive a limited antitumor efficiency due to the insufficient reactive oxygen species (ROS) levels at focal sites and the evolvement of antioxidant defense systems. Herein, we develop an albumin-based nanomedicine to co-deliver chlorin e6 (Ce6) and COH-SR4 (CS), which can simultaneously enhance the yield and lethality of intracellular ROS for amplified photodynamic therapy (PDT). In which, CS acts as both an activator of AMP-activated protein kinase (AMPK) and an inhibitor of glutathione S-transferases (GSTs). Benefiting from it, the prepared HSA-Ce6@COH-SR4 (HCCS) enables positive feedback uptake by promoting AMPK phosphorylation, leading to rapid and extensive tumor accumulation of drugs. As a result, HCCS obviously increases the ROS production to elevate intracellular oxidative stress. Furthermore, HCCS can inhibit GSTs to disturb the antioxidant defense system of tumor cells, intensifying the oxidative damage of ROS. Ultimately, the PDT of HCCS is significantly strengthened by improving the ROS yield and lethality, which greatly declines the proliferation of breast cancer in vivo. This study may open a window in the development of drug co-delivery system for enhanced oxidative therapy of tumors.

Clostridium perfringens phospholipase C, an archetypal bacterial virulence factor, induces the formation of extracellular traps by human neutrophils.

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.

Presynaptic Purinergic Modulation of the Rat Neuro-Muscular Transmission.

ATP, being a well-known universal high-energy compound, plays an important role as a signaling molecule and together with its metabolite adenosine they both attenuate the release of acetylcholine in the neuro-muscular synapse acting through membrane P2 and P1 receptors, respectively. In this work, using a mechanomyographic method, we analyzed the presynaptic mechanisms by which ATP and adenosine can modulate the transduction in the rat m. soleus and m. extensor digitorum longus. N-ethylmaleimide, a G-protein antagonist, prevents the modulating effects of both ATP and adenosine. The action of ATP is abolished by chelerythrin, a specific phospholipase C inhibitor, while the inhibitory effect of adenosine is slightly increased by Rp-cAMPS, an inhibitor of protein kinase A, and by nitrendipine, a blocker of L-type Ca2+ channels. The addition of DPCPX, an A1 receptor antagonist, fully prevents the inhibitory action of adenosine in both muscles. Our data indicate that the inhibitory action of ATP involves metabotropic P2Y receptors and is mediated by phospholipase C dependent processes in rat motor neuron terminals. We suggest that the presynaptic effect of adenosine consists of negative and positive actions. The negative action occurs by stimulation of adenosine A1 receptors while the positive action is associated with the stimulation of adenosine A2A receptors, activation of protein kinase A and opening of L-type calcium channels. The combined mechanism of the modulating action of ATP and adenosine provides fine tuning of the synapse to fast changing conditions in the skeletal muscles.

Short salsalate administration affects cell proliferation, metabolism, and inflammation in polycystic kidney disease

Metabolic reprogramming is a driver of autosomal dominant polycystic kidney disease (ADPKD) progression and a potential therapeutic intervention route. We showed before that the AMP-associated protein kinase (AMPK) activator salsalate attenuates cystic disease progression. Here, we aim to study the early, direct effects of short salsalate treatment in adult-onset conditional Pkd1 deletion mice. Cystic mice were treated with salsalate for two weeks, after which NMR metabolomics and RNA sequencing analyses were performed. Pkd1 deletion resulted in clear metabolomic dysregulation. Short salsalate treatment has small, but significant, effects, reverting acetylcarnitine and phosphocholine concentrations back to wildtype levels, and showing associations with altered purine metabolism. RNA sequencing revealed that short salsalate treatment, next to restoring energy metabolism toward wildtype levels, also affects cell proliferation and inflammation, in PKD. We show that salsalate positively affects major dysregulated processes in ADPKD: energy metabolism, cell proliferation, and inflammation, providing more insights into its working mechanisms.

Effects of HSP70 chaperones Ssa1 and Ssa2 on Ste5 scaffold and the mating mitogen-activated protein kinase (MAPK) pathway in Saccharomyces cerevisiae.

Ste5 is a prototype of scaffold proteins that regulate activation of mitogen-activated protein kinase (MAPK) cascades in all eukaryotes. Ste5 associates with many proteins including Gβγ (Ste4), Ste11 MAPKKK, Ste7 MAPKK, Fus3 and Kss1 MAPKs, Bem1, Cdc24. Here we show that Ste5 also associates with heat shock protein 70 chaperone (Hsp70) Ssa1 and that Ssa1 and its ortholog Ssa2 are together important for Ste5 function and efficient mating responses. The majority of purified overexpressed Ste5 associates with Ssa1. Loss of Ssa1 and Ssa2 has deleterious effects on Ste5 abundance, integrity, and localization particularly when Ste5 is expressed at native levels. The status of Ssa1 and Ssa2 influences Ste5 electrophoresis mobility and formation of high molecular weight species thought to be phosphorylated, ubiquitinylated and aggregated and lower molecular weight fragments. A Ste5 VWA domain mutant with greater propensity to form punctate foci has reduced predicted propensity to bind Ssa1 near the mutation sites and forms more punctate foci when Ssa1 Is overexpressed, supporting a dynamic protein quality control relationship between Ste5 and Ssa1. Loss of Ssa1 and Ssa2 reduces activation of Fus3 and Kss1 MAPKs and FUS1 gene expression and impairs mating shmoo morphogenesis. Surprisingly, ssa1, ssa2, ssa3 and ssa4 single, double and triple mutants can still mate, suggesting compensatory mechanisms exist for folding. Additional analysis suggests Ssa1 is the major Hsp70 chaperone for the mating and invasive growth pathways and reveals several Hsp70-Hsp90 chaperone-network proteins required for mating morphogenesis.

Licochalcone A alleviates abnormal glucolipid metabolism and restores energy homeostasis in diet-induced diabetic mice.

Licochalcone A (LCA) is a bioactive chalcone compound identified in licorice. This study aimed to investigate the effects of LCA on glucolipid metabolism and energy homeostasis, as well as the underlying mechanisms. Blood glucose levels, oral glucose tolerance, serum parameters, and histopathology were examined in high-fat-high-glucose diet (HFD)-induced diabetic mice, with metformin as a positive control. Additionally, changes in key markers related to glucolipid metabolism and mitochondrial function were analyzed to comprehensively assess LCA's effects on metabolism. The results showed that LCA alleviated metabolic abnormalities in HFD-induced diabetic mice, which were manifested by suppression of lipogenesis, promotion of lipolysis, reduction of hepatic steatosis, increase in hepatic glycogenesis, and decrease in gluconeogenesis. In addition, LCA restored energy homeostasis by promoting mitochondrial biogenesis, enhancing mitophagy, and reducing adenosine triphosphate production. Mechanistically, the metabolic benefits of LCA were associated with the downregulation of mammalian target of rapamycin complex 1 and activation of adenosine monophosphate-activated protein kinase, the two central regulators of metabolism. This study demonstrates that LCA can alleviate abnormal glucolipid metabolism and restore energy balance in diet-induced diabetic mice, highlighting its therapeutical potential for the treatment of diabetes.

The NADPH Link between the Renin Angiotensin System and the Antioxidant Mechanisms in Dopaminergic Neurons.

The renin angiotensin system (RAS) has several components including signaling peptides, enzymes, and membrane receptors. The effort in characterizing this system in the periphery has led to the approval of a class of antihypertensives. Much less is known about RAS in the central nervous system. The production of RAS peptides and the expression of several RAS enzymes and receptors in dopaminergic neurons of the substantia nigra has raised expectations in the therapy of Parkinson's disease, a neurodegenerative condition characterized by lack of dopamine in the striatum, the motor control region of the mammalian brain. On the one hand, dopamine production requires reducing power. On the other hand, reducing power is required by mechanisms involved in REDOX homeostasis. This review focuses on the potential role of RAS in the regulation of neuronal/glial expression of glucose-6-phosphate dehydrogenase, which produces the NADPH required for dopamine synthesis and for reactive oxygen species (ROS) detoxification. It is known that transgenic expression of the gene coding for glucose-6-phosphate dehydrogenase prevents the death of dopaminergic nigral neurons. Signaling via angiotensin II G protein-coupled receptors, AT1 or AT2, leads to the activation of protein kinase A and/or protein kinase C that in turn can regulate glucose-6- phosphate dehydrogenase activity, by Ser/Thr phosphorylation/dephosphorylation events. Long-term effects of AT1 or AT2 receptor activation may also impact on the concentration of the enzyme via activation of transcription factors that participate in the regulation of gene expression in neurons (or glia). Future research is needed to determine how the system can be pharmacologically manipulated to increase the availability of NADPH to neurons degenerating in Parkinson's disease and to neuroprotective glia.

SAT269 Orexin Activates Adrenocortical Steroidogenesis With Upregulation Of BMP Signaling By Human H295R Cells

Abstract Disclosure: Y. Soejima: None. N. Iwata: None. R. Nishioka: None. M. Honda: None. Y. Nakano: None. K. Yamamoto: None. A. Suyama: None. F. Otsuka: None. Orexins are neuropeptides that express primarily in the hypothalamus and are composed of two isoforms, orexin A and orexin B. Orexin A binds to orexin receptor type 1 (OX1R) and type 2 (OX2R), while orexin B binds selectively to OX2R. Orexin has been reported to have important roles in various systems including sleep-wake regulation and feeding behavior in the central nervous system, whereas its receptors are also expressed in peripheral tissues including the endocrine system. The expression of orexin receptors and the effects of orexins on adrenocortical steroidogenesis have been shown in experiments using adrenocortical cells. Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor (TGF)-β superfamily, also play functional roles in various endocrine tissues. In our previous experiments, we have revealed that BMP-6 enhances aldosterone production which is induced by angiotensin II through activation of mitogen-activated protein kinase (MAPK) signaling. We have also shown that peripheral clock genes have functional roles in modulating adrenocortical steroidogenesis by interacting with adrenocortical BMP system. However, the molecular mechanism of physiological effects of orexin and its interaction with BMP signaling remains unclear. In the present study, we investigated the effects of orexin on adrenal steroidogenesis using human adrenocortical H295R cells, by focusing on the interaction with adrenocortical BMP signaling. First, it was confirmed that both OX1R and OX2R were expressed in H295R cells. It was revealed that orexin A (10-300 nM) stimulation for 24 h directly increased mRNA levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily B member 2 (CYP11B2) and cytochrome P450 family 17 subfamily A member 1 (CYP17A1) in a concentration-dependent manner. Orexin A further upregulated the expression of these steroidogenic enzymes in H295R cells co-treated with forskolin (FSK). Next, the interrelationship between orexin and BMP signaling was examined. Stimulation with BMP-6 readily activated Smad1/5/9 phosphorylation in H295R cells and the pretreatment with orexin A for 24 h enhanced the Smad1/5/9 phosphorylation induced by BMP-6. On the other hand, BMP-6 significantly suppressed the expression of OX1R but not that of OX2R. In our previous study, we have reported that BMP-6 upregulates the adrenocortical steroidogenesis via the IP3 and MAPK signaling in H295R cells, suggesting that orexin may have a pivotal role as a functional modulator of BMP-6. Collectively, it was revealed that orexin enhances adrenocortical steroidogenesis by regulating BMP signaling with a feedback system in human adrenocortical cells. Here, we will also discuss the relationship with BMP receptors and the effects on endogenous BMP expression. Presentation: Saturday, June 17, 2023

Modulation of Structure and Dynamics of Cardiac Troponin by Phosphorylation and Mutations Revealed by Molecular Dynamics Simulations.

Adrenaline acts on β1 receptors in the heart muscle to enhance contractility, increase the heart rate, and increase the rate of relaxation (lusitropy) via activation of the cyclic AMP-dependent protein kinase, PKA. Phosphorylation of serines 22 and 23 in the N-terminal peptide of cardiac troponin I is responsible for lusitropy. Mutations associated with cardiomyopathy suppress the phosphorylation-dependent change. Key parts of troponin responsible for this modulatory system are disordered and cannot be resolved by conventional structural approaches. We performed all-atom molecular dynamics simulations (5 × 1.5 μs runs) of the troponin core (419 amino acids) in the presence of Ca2+ in the bisphosphorylated and unphosphorylated states for both wild-type troponin and the troponin C (cTnC) G159D mutant. PKA phosphorylation affects troponin dynamics. There is significant rigidification of the structure involving rearrangement of the cTnI(1-33)-cTnC interaction and changes in the distribution of the cTnC helix A/B angle, troponin I (cTnI) switch peptide (149-164) docking, and the angle between the regulatory head and ITC arm domains. The familial dilated cardiomyopathy cTnC G159D mutation whose Ca2+ sensitivity is not modulated by cTnI phosphorylation exhibits a structure inherently more rigid than the wild type, with phosphorylation reversing the direction of all metrics relative to the wild type.

Schistosoma mansoni excretory-secretory products induce protein kinase signalling, hyperkinesia, and stem cell proliferation in the opposite sex

Adult male and female schistosomes in copula dwell within human blood vessels and lay eggs that cause the major Neglected Tropical Disease human schistosomiasis. How males and females communicate to each other is poorly understood; however, male-female physical interaction is known to be important. Here, we investigate whether excretory-secretory products (ESPs), released into the external milieu by mature Schistosoma mansoni, might induce responses in the opposite sex. We demonstrate that ESPs adhere to the surface of opposite sex worms inducing the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways, particularly in the parasite tegument. Furthermore, we show that mature worms stimulated signalling in juvenile worms. Strikingly, we demonstrate that ESPs from the opposite sex promote stem cell proliferation, in an ERK- and p38 MAPK-dependent manner, in the tegument and within the testes of males, and the ovaries and vitellaria of females. Hyperkinesia also occurs following opposite sex ESP exposure. Our findings support the hypothesis that male and female schistosomes may communicate over distance to modulate key processes underlying worm development and disease progression, opening unique avenues for schistosomiasis control.

Siraitia grosvenorii Extract Attenuates Airway Inflammation in a Mouse Model of Respiratory Disease Induced by Particulate Matter 10 Plus Diesel Exhaust Particles.

Exposure to particulate matter (PM) causes considerable breathing-related health risks. Siraitia grosvenorii fruit is a traditional remedial plant used in Korea and China to treat respiratory diseases. Our recently published study showed that S. grosvenorii extract (SGE) ameliorated airway inflammation in lipopolysaccharide- and cigarette-smoke-induced chronic obstructive pulmonary disease in mice. Thus, we aimed to assess the inhibitory effects of SGE on airway inflammation in mice exposed to a fine dust mixture of PM10 (PM diameter < 10 mm) and diesel exhaust particles (DEPs) known as PM10D. The mice (BALB/c) were treated with PM10D via intranasal injection three times over a period of 12 days, and SGE 70% ethanolic extract (50 or 100 mg/kg) was orally administered daily for 12 days. SGE attenuated neutrophil accumulation and the number of immune B and T cells from the lung tissue and bronchoalveolar lavage fluid (BALF) of the PM10D-exposed mice. SGE reduced the secretion of cytokines and chemokines, including interleukin (IL)-1α, tumor necrosis factor (TNF)-α, IL-17, C-X-C motif chemokine ligand (CXCL)1, and macrophage inflammatory protein (MIP)-2 in the BALF. Airway inflammation, infiltration of inflammatory cells, and collagen fibrosis in the lung after PM10D exposure were investigated via histopathological analysis, and SGE treatment ameliorated these symptoms. SGE decreased the mRNA expression of mucin 5AC (MUC5AC), CXCL1, TNF-α, MIP-2, and transient receptor potential ion channels in the lung tissues. Furthermore, SGE ameliorated the activation of mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) signaling by PM10D in the lungs. We conclude that SGE attenuated PM10D-induced neutrophilic airway inflammation by inhibiting MAPK/NF-κB activation. These results show that SGE may be a candidate for the treatment of inflammatory respiratory diseases.

Исследование роли нейротрофических факторов в регуляции регенерационных процессов в поврежденных соматических нервах при действии пептидного препарата «Семакс»

The content of neurotrophic factors in damaged somatic nerves under the action of the drug «Semax» was studied and their role in the regulation of regenerative processes in injured nerve conductors was established. It has been shown that intramuscular administration of the drug is accompanied by a significant increase in the level of NGF and NT-3 both in the proximal and distal parts of the nerve. At the same time, there are no significant changes in the quantitative content of neuroregulin-1 against the background of its use. The data obtained suggest that one of the mechanisms of action of Semax is its ability to interact with Schwann cells and stimulate the release of NGF and NT-3, which facilitate the regeneration of damaged axons and do not affect the synthesis of neuroregulin-1. In addition, the study of the quantitative content of individual protein fractions showed that the drug «Semax» has the most pronounced effect on the level of neurofilament-H in both segments of the nerve conductor, which indicates the important role and activation of the mitogen-activated protein kinase (MAPK / ERK) signalling pathway, which regulates processes of cytoskeletal protein synthesis and axon growth. Nevertheless, it was shown that in the variant of the experiment using Semax, there was a decrease in the level of GAP-43, which is a key marker of axonal growth, both in the proximal and distal segments of the nerve. The data obtained most likely indicate that the intramuscular administration of the drug does not affect the processes of axon growth, but is aimed at maintaining the survival of neurons and accelerated restoration of the functional state of nerve fibres, which is also confirmed by the appearance of an action potential and the ability of the nerve to conduct it against the background of the use of the Semax drug.

Anti-Photoaging Effect of Jawoongo via Regulating Nrf2/ARE and TGF-β/Smad Signaling in In Vitro Photoaging Model

Jawoongo is a traditional prescription used widely in East Asia for external applications and is effective for burns, sunburn, wounds, and symptoms of dryness in skin disease. This study was established to examine how Jawoongo treatment affects the photodamage of HaCaT cells caused by ultraviolet B (UVB) irradiation and explore its possible anti-photoaging mechanisms from the perspective of the associated signaling pathways. An in vitro photoaging model was established by irradiating HaCaT with UVB, and the expression of photodamage markers such as matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) was assessed by enzyme-linked immunosorbent assay (ELISA), FACs, and RT-PCR. Western blotting was also used to ascertain the expression levels of the TGF-β/Smad and nuclear erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) signaling pathways. The findings of this study showed that the increased production of ROS, MMP-1, and MMP-3 due to UVB irradiation was reduced by Jawoongo. In addition, the Nrf2/ARE and TGF-β/Smad pathways were upregulated by Jawoongo, while it reduced phosphorylation activation of the mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) pathway. This study shows that Jawoongo regulates the Nrf2/ARE and TGF-β/Smad signaling pathways in UVB-irradiated HaCaT cells, highlighting its potential to protect against and repair skin photodamage.